1. Field of the Invention
This invention relates to the use of certain triterpenes which are inhibitors of nuclear factor kappa B (NF-κB). In particular, it relates to useful triterpenes and pharmaceutical compositions containing them for use in the treatment of inflammatory diseases and for cancers susceptible to an NF-κB inhibitor. The present invention also relates to compounds and methods useful to inhibit cell proliferation and for the induction of apoptosis.
2. Description of the Related Art
Certain triterpenes have previously been known to inhibit the activity of the endothelin receptor by acting as a receptor antagonist. In U.S. Pat. No. 5,587,505 to Konoike issued Dec. 24, 1996, U.S. Pat. No. 5,463,107 to Konoike issued Oct. 31, 1995 and U.S. Pat. No. 5,248,807 to Fujimoto issued Sep. 28, 1993 there are described certain triterpenes active as an antagonist against the endothelin receptor. These compounds are described as useful in treatment of disease states that are caused by excessive secretion of endothelin. These compounds are further shown to be a competitor of endothelin for binding to the endothelin receptor. No further activity or use is described.
Endothelin is a vasoconstrictor peptide composed of 21 amino acids and derived in mammals from the endothelium. These endothelin receptors exist in various tissue and organs such as vessels, trachea and the like and their excessive stimulation can lead to circulatory diseases such as pulmonary hypertension, acute and chronic heart failure, acute and chronic renal failure, atherosclerosis, cerebrovascular diseases and the like.
NF-κB is one of the principal inducible transcription factors in mammals and has been shown to play a pivotal role in the mammalian innate immune response and chronic inflammatory conditions (Jour. Pharm. and Phar. 2002, 54: 453-472). The signaling mechanism of NF-κB involves an integrated sequence of protein-regulated steps and many are potential key targets for intervention in treating certain NF-κB cascade dependant inflammatory conditions and cancers.
More specifically, the family of NF-κB transcription factors comprises important regulatory proteins that impact virtually every feature of cellular adaptation, including responses to stress, inflammatory reactions, activation of immune cell function, cellular proliferation, programmed cell death (apoptosis), differentiation and oncogenesis (1). NF-κB regulates more than 150 genes, including cytokines, chemokines, cell adhesion molecules, and growth factors (2). It is therefore not surprising that diseases result when NF-κB-dependent transcription is not appropriately-regulated. NF-κB has been implicated in several pathologies, including certain cancers (e.g., Hodgkin's disease, breast cancer, and prostate cancer), diseases associated with inflammation (e.g., rheumatoid arthritis, asthma, inflammatory bowel disease (e.g., Crohn's disease and ulcerative colitis), alcoholic liver disease, non-alcoholic steatohepatitis, pancreatitis, primary dysmenorrhea, psoriasis, and atherosclerosis) and Alzheimer's disease. Several mediators of inflammation are under the influence of activated NF-κB including inducible nitric oxide synthase, the subsequent production of nitric oxide and prostaglandin synthase. It has further been shown that compounds which interfere with COX-2 act via the inhibition of NF-κB. NF-κB consists of different combinations of Rel proteins in various heterodimers and homodimers and has previously been represented by the subunits p65/p50. All the Rel proteins share a conserved region of 300 amino acids at the N-terminal responsible for DNA-binding, dimerisation and interaction with the NF-κB inhibitory protein I-kappaB. NF-κB is responsible in several signaling cascades and the two most important of which are ones associated with mammalian immune response of the interleukin/lipopolysaccharide pathway. There are pathways involved with NF-κB that are critically involved in apoptosis. NF-κB binding by RelA is constituitively elevated in human metastatic melanoma cultures relative to normal melanocytes.
NF-κB is a collective name for dimeric transcription factors comprising the Rel family of DNA-binding proteins (3, 4). All members of this family are characterized by the presence of a conserved protein motif called the Rel homology domain (RHD) that is responsible for dimer formation, nuclear translocation, sequence-specific DNA recognition and interaction with inhibitory proteins collectively known as I-κB. Any homodimer or heterodimer combination of family members constitutes NF-κB.
Regulation of NF-κB Activity
The activity of NF-κB is regulated through an assortment of complex signaling pathways. NF-κB is negatively regulated through interaction with I-κB (5). Each I-κB possesses an N-terminal regulatory domain for a signal dependent I-κB proteolysis, a domain composed of six or seven ankyrin repeats to mediate interaction with the RelI proteins, and a C-terminal domain containing a PEST motif that is implicated in constitutive I-κB turnover. Inactive forms of NF-κB reside in the cytoplasm as NF-κB/I-κB complexes, because I-κB binding to NF-κB blocks the ability of the nuclear import proteins to recognize and bind to the nuclear localization signal in the RHD.
NF-κB activation occurs when NF-κB is translocated to the nucleus following its release from I-κB. I-κB dissociation arises through its phosphorylation by an inducible I-κB kinase (IKK) and ubiquitination by I-κB ubiquitin ligase, which flags it for proteolysis by the 26S proteosome. Since the ubiquitin ligase and the 26S proteosome are constitutively expressed, the de-repression of NF-κB functional activity is largely governed by those signals that induce the expression of IKK, which include inflammatory cytokines, mitogens, viral proteins, and stress.
IKK is also known as the signalsome, which consists of a large multi-subunit complex containing the catalytic subunits IKK.alpha./IKK-1 and IKK.beta./IKK-2, a structural subunit termed NF-κB essential modulator (NEMO), as well as perhaps other components (6, 7). NEMO, also known as IKK.γ. and IKKAP-1, functions as an adapter protein to permit communication between the catalytic subunits and upstream activators (7). Activation of NF-κB is a tightly controlled process and cannot occur without NEMO (8, 9).
Protein phosphorylation positively regulates NF-κB activity (1). Protein phosphorylation enhances the transcriptional activity of NF-κB, presumably through the phosphorylated protein's interaction with other transcriptional co-activators. Protein kinase A (PKA), casein kinase II (CKII), and p38 mitogen-activated protein kinase (MAPK) have been implicated in the phosphorylation of NF-κB.
The activity of NF-κB is also subject to autoregulatory mechanisms to ensure that NF-κB-dependent transcription is coordinately-linked to the signal-inducing response. For example, the I-κB genes contain NF-κB binding sites within their promoter structures that result in their increased transcription upon NF-κB binding. The expressed I-κB proteins migrate into the nucleus to bind the NF-κB and mediate transport of NF-κB to the cytoplasm where it remains inactive.
Role of NF-κB in Disease and Disorders
NF-κB contributes to progression of cancers by serving both as positive regulators of cell growth and as a negative regulator of apoptosis (10, 11). NF-κB stimulates expression of cell cycle-specific proteins c-Myc and cyclin D1 (12, 13). The constitutive expression of these proteins results in sustained cell proliferation. Continued expression of c-Myc ultimately leads to apoptosis. NF-κB can block c-Myc's apoptosis effects, thereby stimulating proliferation without cytotoxicity. NF-κB also inhibits the ability of Tumor Necrosis Factor (TNF) to induce cell death as well as protect cells from the effects of ionizing radiation and chemotherapeutic drugs (14). Thus, NF-κB promotes both hyperplasia and resistance to oncological treatments, which are hallmarks of many cancers.
Inhibition of NF-κB activation has been linked to the chemopreventive properties of several anti-cancer compounds (e.g., selenium, flavonoids, etc.) (15, 16). Although long-term inhibition could have unwanted effects on immune response, down-regulation of NF-κB activity is considered a very attractive strategy for developing new cancer treatments.
Recently, Shen et al. demonstrated that certain oligonucleotides that contain polyguanonsines are potent inhibitors of the proliferation of murine prostate cancer cells (17). The specific DNA-binding activities of NF-κB and another transcription factor, AP-1 were reduced in cells treated with these oligonucleotides. Oligonucleotides displaying antiproliferative effects were capable of forming higher order structures containing guanosine-quartets (G-quartets). The requirement of G-quartets for inducing apoptosis was suggested by experimental observations wherein mutations that destroyed the capacity to form a G-quartet structure correlated with abolishment of the antitumor activities of the oligonucleotide (17).
In the case of inflammation, NF-κB plays important roles in both the initiation and maintenance of the inflammatory response (1). Activated T cells, such as activated CD4+ T helper cells, trigger immune inflammation. The T helper cell population can differentiate further to two subset populations that have opposite effects on the inflammatory response. The Th1 subset is considered proinflammatory, as these cells mediate cellular immunity and activate macrophages. The Th2 subset is considered anti-inflammatory, as these cells mediate humoral immunity and down-regulate macrophage activation. The subsets are distinguishable by the different types of cytokine profiles that they express upon differentiation. NF-κB stimulates production of cytokine profiles characteristic of the Th1 subset type, leading to a proinflammatory response. Conversely, suppression of NF-κB activation leads to production of cytokine profiles characteristic of the Th2 subset type that mediates an anti-inflammatory response.
Once activated, these inflammatory cytokines and growth factors can act through autocrine loops to maintain NF-κB activation in non-immune cells within the lesion (1). For example, NF-κB regulates the expression of cytokines Interleukin 1 β (IL-1 β) and Tumor Necrosis Factor alpha (TNFα), which are considered essential mediators of the inflammatory response. Conversely, these gene products positively activate NF-κB expression that leads to persistence of the inflammatory state. For example, TNF products have been implicated in promoting inflammation in several gastrointestinal clinical disorders that include: alcoholic liver disease, non-alcoholic steatohepatitis, prancreatitis (including chronic, acute and alcohol-induced), and inflammatory bowel disorders, such as ulcerative colitis and Crohn's Disease.
Continued NF-κB activation also promotes tissue remodeling in the inflammatory lesions (1). Several NF-κB-responsive genes have been implicated in this regard and include growth factors that are important to neovascularization (e.g., VEGF), matrix proteinases (including metalloproteases), cyclooxygenase, nitric oxide synthase, and enzymes that are involved in the synthesis of proinflammatory prostaglandins, nitric oxide, and nitric oxide metabolites (1). Such tissue remodeling is often accompanied by breakdown of healthy cells as well as by hyperplasia, both of which are often observed in rheumatoid arthritis and other inflammatory diseases (1).
Suppression of NF-κB activity alleviates many inflammatory disease conditions and increases the susceptibility of certain cancers to effective treatment. Several anti-inflammatory drugs directly target the NF-κB signaling pathway. Glucocorticoids, one member of the general steroid family of anti-inflammatory drugs, interfere with NF-κB function through the interaction of the glucocorticoid receptor with NF-κB (18). Gold compounds interfere with the DNA-binding activity of NF-κB (19). Aspirin and sodium salicylate, as representatives of non-steroid anti-inflammatory drugs, inhibit IKKβ activity and thereby prevent signal-inducible I-κB turnover (20). Dietary supplements with anti-inflammatory and anti-tumor activities prevent NF-κB activation by interfering with pathways leading to IKK activation. Vitamins C and E, prostaglandins, and other antioxidants, scavenge reactive oxygen species that are required for NF-κB activation (21, 22). Specific NF-κB decoys that mimic natural NF-κB ligands (e.g., synthetic double-stranded oligodeoxynucleotides that contain the NF-κB binding site) can suppress NF-κB activity and prevent recurrent arthritis in animal models (23).
Despite the promise of anti-inflammatory drugs in treating inflammatory diseases, many diseases are non-responsive to these modalities. For example, many patients with chronic inflammatory diseases, such as Crohn's disease, fail to respond to steroid treatment. Recent studies suggest that one basis for the steroid unresponsiveness may be attributed to NF-κB and other NF-κB-responsive gene products antagonizing glucocorticoid receptor expression, which is necessary for the steroid's anti-inflammatory activity (24).
Alzheimer's disease represents another example of a condition that displays an inflammatory component in its pathogenesis. Recent studies indicate that abnormal regulation of the NF-κB pathway may be central to the pathogenesis of Alzheimer's disease. NF-κB activation correlates with the initiation of neuritic plaques and neuronal apoptosis during the early phases of the disease. For example, NF-κB immunoreactivity is found predominantly in and around early neuritic plaque types, whereas mature plaque types display reduced NF-κB activity (25).
Numerous plant-derived products have been identified as inhibitors of NF-κB activation and a large number of pure compounds have been shown to interfere with the cascade leading to NF-κB activation. While these compositions are clearly useful to date and many agents which inhibit NF-κB also inhibit AP-1 in certain U937 cells, no compositions which also inhibit endothelin have been discovered.
Accordingly it would be extremely useful to find compositions which can inhibit both endothelin and NF-κB.